New and biologically active diphenylmethane derivatives and method of making the same



Patented June 29, 1954 NEW AND BIOLOGICALLY ACTIVE DI- PHENYLMETHANE DERIVATIVES AND METHOD OF MAKING THE SAME Ladislaus Arthur Hahn, Malmo, Sweden, and

.Ianos Fekete, Lorena, Brazil, assignors to Aktiebolaget Ferrosan, Malmo, Sweden, a corporation of Sweden No Drawing. Application September 9, 1952, Serial No. 308,701

Claims priority, application Sweden December 9, 1949 1 Claim.

This application is a continuation-in-part of our co-pending application No. 199,564, filed- December 6, 1950, now abandoned.

The present invention relates to new biologically active diphenylmethane derivatives and to a method of making the same.

An enzyme which is capable of splitting mucopolysaccharides and amongst them particularly the hyaluronic acid has been discovered i. a. in the testicles and the sperm of mammals, furthermore in the leech, in serpents and insects poisons and in certain bacteria. The enzyme has been denominated mucinase but the name has later been changed to hyaluronidase, or mucopolysaccharase, respectively.

The substrate hyaluronic acid is rather common in the animal organism. Among the most important sources may be mentioned the interfibrillar substance of mesenchymal tissues, especially of the connective tissues, furthermore the vitreous body of the eye, the synovia and the gel that surrounds the ovum on its way through the oviduct. Another important substrate for the hyaluronidase, the chondroitin sulfuric acid, is a substantial component of all sorts of cartilage, thus also of the cartilage of the joints.

The splitting efiect of the hyaluronidase on the hyaluronic acid has been elucidated to a large extent: the highly polymeric acid is depolymerized and split into smaller units, the glucosidic bonds between the elements of the hyaluronic acid, i. e. the glucuronic acid and the N-acetyl glucos amine, being hydrolyzed. In the complete splitting up of the hyaluronic acid to monosaccharide units also other enzymes are active. One of these enzymes has been isolated from bulls testicles and has been denominated mucooligosaccharase. The splitting of the hyaluronic acid and of other mucopolysaccharides, for instance the chondroitin sulfuric acid, through hyaluronidase may be experimentally shown and quantitatively determined for instance (a) by observing the decrease in viscosity of the substrate or (b). by determining the reducing groups which are liberated at the hydrolysis. There is also a biological method of determination which is based upon the influence of the hyaluronidase on the permeability of the skin, said influence being explained in the following.

The effect of the hyaluronidase in vivo corresponds to its effect on mucopolysacc-harides in vitro: for instance the hyaluronic acid of the skin is split by the action of hyaluronidase, which is brought into the tissue in one way or other, and. loses its high viscosity. This causes the skin to become much more permeable. This increase of the permeability is rather general and results therein, that as well physiological as non-physiological substances, such as water, salts, added chemical compounds of all kinds, coloring matters, poisons, bacteria and virus will spread many times quicker in the tissue if hyaluronidase is present. This may easily be shown experimentally by intracutaneous injection of 0.2 millilitre of Indian ink for instance on one side of the back of a rabbit, the same amount of ink to which there has been added some micrograms of hyaluronidasebeing injected on the other side. The ink to which no hyaluronidase has been added spreads only very slowly in the skin. The ink which has been mixed with the enzyme spreads in a few minutes over an area of 200 0111. or more. That which has been said about the eiiect of the hyaluronidase upon the permeability of the skin holds true also in respect of other kinds of mesenchymal tissue.

The high viscosity of the synovial fluid depends to a great extent on the hyaluronic acid and the same holds true i. a. of the vitreous body of the eye and of the gel that surrounds the ovum in the oviduct. The effect of the hyaluronidase upon such materials is to make them lose their viscosity so that they become fluent.

The hyaluronidase of certain bacteria is of great importance for various reasons. Some of the pathogenic bacteria containing hyaluronidase, such as streptococci, are connected with certain types of rheumatic diseases. Since the viscosity of the synovial fluid depends mainly on its content of hyaluronic acid and since the cartilage of the joints contains chondroitin sulfuric acid it was assumed that at least some forms of rheumatic and other joint diseases are caused directly or indirectly by hyaluronidase.

The effect of the hyaluronidase upon connective tissues results therein that bacteria containing or producing this enzyme will penetrate through the organism much easier than other bacteria so that the risk of infection is highly increased. The hyaluronidase-bearing bacteria need not necessarily be pathogenic themselves, or they may be pathogenic only to a small degreenevertheless they may mean a great danger to the organism by facilitating the penetration of other non-physiological substances, such as other bacteria, virus etc., into tissues and organs. This is of particular importance since many highly pathogenic virus show a rather low penetration capability. If the organism infected by hyaluronidase-producing bacteria infection with the said agents may result.

Since hyaluronic acid. is present also in cap- 3 illary walls the capillary permeability is influenced by hyaluronidase. The hyaluronidase may therefore promote or facilitate the penetration of infective substances through the capillaries.

Certain substances inhibiting the effect of the hyaluronidase are known. As an example carboxy-p-benzoquinone, rutin, ascorbic acid, heparin, hexylresorcinol, certain sera fractions and nitrates of hyaluronic acid may be mentioned.

It has now been discovered that an inhibition of the action of hyaluronidase which is many times stronger than that of the above-mentioned known substances is developed by certain diphenylmethane derivatives. These derivatives are obtained by condensing with formaldehyde dihydroxy benzoic acids selected from the group consisting of 2.3-, 2.5-, 2.6-, 3 .4- and 3.5-dihydroxy benzoic acid. The preparation of compounds of similar type such as methylene-disalicylic acid and methylene-digallic acid has been described before. We investigated the hyaluronidase inhibiting power of these compounds but found them to be only weakly active. Expressed in relative units the inhibitory power of methylene-disalicyclic acid is 30 and that of methylene-digallic acid 80. Earlier described inhibitors, for example hexylresorcinol, show inhibitory activities up to 100 relative units. The compounds here described show inhibitory activities of 3000 relative units and more. This very high inhibitory activity on hyaluronidase makes it possible to use these compounds in the treatment of rheumatoid arthritis on one side and infectious diseases induced by hyaluronidase producing bacteria on the other side.

The inhibitors according to the invention may also be introduced into the organism in a suitable manner, as per os, by superficial treatment or by subcutaneous, intramuscular or intraperitoneal injection etc. in order to prevent the spreading effect of the hyaluronidase in poisonings and infections. As mentioned above serpents and insects poisons and various pathogenic bacteria contain hyaluronidase and in part also other mucopolysaccharases. If these enzymes get into the organism such as by biting or infection they hydrolyse the hyaluronic acid of the skin and other tissues so that the poison or the bacteria are more easily infiltrated in the organism. Also infection with other infective substances which are free from hyaluronidase, such as bacteria and virus, spreads much quicker in the organism if the sameis infected with hyaluronidase-bearing bacteria. The inhibitors according to this invention inhibit the action of the hyaluronidase of poisons and bacteria upon tissues and thus prevent or limit infection. The action diilers principally from the treatment with anti-toxins or anti-sera (no specificity).

The compounds which are the matter of the present invention are prepared in the following manner:

The dihydroxybenzoic acid is dissolved or suspended in a suitable solvent (for instance water, alcohol, dioxan, ether, acetic acid, hydrochloric acid, sulfuric acid). Then the formaldehyde solution is added either all at one time or successively in the course of the reaction. The

latter mode of operation is used in certain cases in order to control the condensation reaction.

Sometimes the condensation will occur already at room temperature and in absence of condensation agents. However, in most cases it is preferable, or necessary, that a. condensation agent is present. The reaction then occurs either at room temperature, or at an elevated temperature, and the reaction period may vary from some minutes up to several hours depending upon the reactive power of the compound in question, the concentration of the condensing agent and other conditions. As condensation agents mineral acids (for instance hydrochloric acid, sulfuric acid) or alkalis (for instance alkali hydroxides) may be used.

If the starting material is sensitive to air oxygen the reaction should preferably be carried out in an inert atmosphere. Since the reaction mostly takes place in a heterogeneous phase the reaction mix should be vigorously stirred.

As generally the reaction product is considerably less soluble than the starting material, purification of the reaction product may often be carried out taking advantage of the said differenc in solubility.

The production of certain of the contemplated substances may sometimes be carried out in such a manner that the carboxylic groups are introduced after the condensation has been eiiected.

The product of the condensation of the dihydroxy benzoic acids mentioned with formaldehyde under conditions here described was found to be a mixture of simple diphenylmethane derivatives and their polycondensation products. The proportion between the yield of diphenylmethane derivatives and of that of polycondensed products depends on (a) th position of the OH-groups in the benzene ring and (b) the conditions used. For example 2.6-dihydroxy benzoic acid has a pronounced tendency to the building of polycondensed products. The presence of condensing agents, increasing concentration of mineral acids, increasing temperature and increasing time of reaction generally favour the production of polycondensed products. The separation of the simple diphenylmethane derivatives from the corresponding polycondensed products is rather difiicult. Fractionation with organic solvent, for example alcohol, gives in some cases good results.

We found that also the polycondensed products mentioned above show very pronounced inhibitory action on hyaluronidase. In most cases the inhibitory power of the polyconsensed product is somewhat higher than the inhibitory power of the corresponding diphenylmethane derivative.

Determination of the inhibiting power in vitro The following method has been used for determining the inhibiting power of the various compounds produced: in an Ostwald viscosimeter the viscosity of a 0.1% hyaluronic acid dissolved in an isotonic phosphate buffer (pl-I7) was determined at 37 C. To this solution there was added a certain amount of hyaluronidase solution (in phosphate buffer, pH '7) produced from bulls testicles. Through repeated viscosity determinations of the hyaluronic acid-dyaluronidase-solution it is possible to draw a diagram with the values thus obtained. The time elapsed after the addition of the enzyme solution is plotted against the abscissa, and against the ordinate are plotted the various outflow periods for the hyaluronic acid-hyaluronidase-solution. In this way a standard curve is obtained express ing the rate of decomposition of the hyaluronic acid. In this diagram the so-called half-period may be determined, i. e. the period it takes for the solution to reach the half, of the viscosity value of the hyaluronic acid. The half-period is the point of section of the decomposition curve with a line (parallel to the abscissa) expressing the half of the viscosity of the pure hyaluronic acid.

For determining th inhibiting power of a compound the hyaluronidase-enzyme-solution is mixed with a certain amount of an inhibitor solution of known concentration (dissolved in water and with pH adjusted at 7). The mixture of hyaluronidase and inhibitor is first allowed to stand for 30 minutes in a water thermostat at 37 C. whereupon the same series of viscosity determinations is carried out as when plotting the standard curve. In case of inhibition the half-period will be longer than according to the standard curve. A relative inhibition value is thus obtained by dividing the new half-period by the standard half-period. The inhibiting power may advantageously be expressed as the reciprocal value of the concentration of the inhibitor solution in which concentration the inhibitor in question causes a fivefold prolongation of the half-period.

In order to eliminate possible differences in the quality of the hyaluronic acid and the hyaluronidase, respectively, salicylic acid was used as a standard inhibitor. All inhibition factors will thus be relative figures expressing how many times more effective the inhibitor in question is than salicylic acid.

Determination of the inhibiting power in vivo In vivo tests are advantageously carried out on rabbits. The hyaluronidase is injected intravenously into the animals. The action of the enzyme may be demonstrated through injection of Indian ink or methylene blue (the dye stuffs being injected intracutaneously in the shaved back of the rabbit). The more the dye stuff is spread in the skin, th more active the enzyme may be said to be. The effect of the enzyme is characterized also by a decreased elasticity of the skin of the rabbit. Finally the effect of the enzyme may be demonstrated as a shortened resorption period for an intracutaneously injected salt solution.

If, prior to the injection of the enzyme, the rabbits ar given an intramuscular injection of inhibitor solution, the effect of the enzyme either wholly disappears or appears only in very small degree.

Example 1 2,2,5,5' tetrahydroxy 3,3 dicarboxy diphenylmethane (methylene digentisic acid) may be produced in the following manner:

In 90 grams of cold 50% sulfuric acid grams of gentisic acid are suspended whereupon 4 grams of a 40% formaldehyde solution are added. The reaction mix is boiled during 5 hours with vigorous agitation. The warm product is filtered through a glass filter and the precipitate is pulverized and boiled with water, whereupon the same is washed several times with hot water. The substance is easily soluble in alcohol, acetone and alkalis, but very little soluble in water. Its decomposition point is above 260 C. Relative hyaluronidase inhibitory power: 4000 (salicylic acid=1).

Example 2 3,3',4,4 tetrahydroxy 6,6 dicarboxy diphenylmethane may be produced by condensing 2 molar weights of 3,4-dihydroxy benzoic acid 6 and 1 molar weight of formaldehyde according to Example 1. Relative inhibitory power: 3200.

Example 3 3,3,4,4'-tetrahydroxy-5,5-dicarboxy diphenylmethane maybe produced by condensing 2 molar weights of 2,3-dihydroxy benzoic acid with 1 molar weight of formaldehyde in the presence of 10% by weight of hydrochloric acid. The reaction mix is heated during 3 hours on a boiling water bath. The reaction product may be isolated in the same manner as in Example 1. Relative inhibitory power: 3100.

Example 4 2,2AA;-tetrahydroxy-3,3-dicarboxy diphenylmethane may be produced by condensin 2 molar weights of 2,6-dihydroxy benzoic acid with 1 molar weight of formaldehyde in the presence of 10% by weight of hydrochloric acid in a carbon dioxide atmosphere. The reaction mix is heated during 3 hours on a boiling water bath. The reaction product may be isolated in the same manner as in Example 1. Relative inhibitory power: 3400.

Example 5 Example 6 In 100 grains of cold sulfuric acid 20.8 grams of gentisic acid are suspended and then 3.75 grams of 40% formaldehyde are added. This mixture is heated on a boilin water bath during 12 hours. The reaction product is filtered oif and washed with hot water until free from sulfuric acid. 19.5 grams of a dark green product were obtained. Its decomposition point is above 300 C. Relative inhibitory power: 4000. This I product consists in part of 2.2'.5.5-tetrahydroxy- 3.3'dicarboxy diphenylmethane (methylenedigentisic acid) and in part of polycondensed products of fairly high molecular size. Any exact formula for the latter cannot be given. Methylenedi-gentisic acid is easily soluble in alcohol and can be purified by extraction of the condensation product with a little volume of alcohol and reprecipitation with water.

Example 7 In 100 grams of cold 75% sulfuric acid 15.4 grams of 3.4-dihydroxy-benzoic acid are suspended and then 3.75 grams of 40% formaldehyde are added. This mixture is heated on a boiling water bath during 12 hours. The reaction product is filtered off and washed with hot water until free from sulfuric acid. 13.7 grams of a dark brown product were obtained. Decomposition point above 280 C. This product is a mixture of methylene-di-protocatechuic acid and of polycondensed products. Relative inhibitory power: 3750.

Example 8 In a mixture of grams of 60% sulfuric acid and 40 grams of acetic acid 15.4 grams of 2.3-dihydroxy-benzoic acid are dissolved. The solution is warmed on a, boiling water bath and 4 grams of a 40% formaldehyde solution are added in the course of half an hour. Heating is continued during 8 hours. The warm product is filtered through a glass filter and the precipitation is pulverized and washed several times with hot water. Yield: 11.9 grams. Relative inhibitory power: 3400.

Example 9 In 120 grams of cold 50% sulfuric acid 20.8 grams of 2.6-dihydroxy benzoic acid are suspended. 3.75 grams of 40% formaldehyde solution are then added and the mixture is heated on a boiling water bath during 3 hours. The condensation product obtained is washed with hot water and dried in vacuum. Yield: 18.0 grams. Relative inhibitory power: 4200.

Example 10 In 80 grams of diluted hydrochloric acid (1:1) 18.1 grams of 3.5-dihydroxy benzoic acid are suspended. Aiter the addition of 4 grams of 40% formaldehyde solution the mixture is boiled during 12 hours. The reaction product is isolated in the same manner as in Example 1. Relative inhibitory power: 3250.

Example 11 In 800 grams of 50% sulfuric acid 110 grams of hydroquinone are dissolved. After addition of 37.5 grams of 40% formaldehyde solution the mixture is boiled during 2 hours and the condensation product separated in the same manner as in Example 1. Yield: 108 grams of methylenedihydroquinone (including polycondensed products). This product is suspended in 10 times its quantity of a 50% sodium bicarbonate solution and is heated during 4 hours on a boilin water bath in a carbondioxid atmosphere. By neutralizin with hydrochloric acid a precipitate is obtained which is purified by two reprecipitations. The product obtained is methylenedi-gentisic acid containing some polycondensed products built up of methylenedi-gentisic acid units. Relative inhlbitory activity 4600.

Example 12 In 600 grams of 10% by weight of hydrochloric acid grams of pyrocatechol are dissolved. 4 grams of 40% formaldehyde solution are added. The condensation and the purification of the reaction product are carried out in the same manner as in Example 3. Yield: 105 grams of methylenedi pyrocatechol (including polycondensed products). 100 grams of this substance are mixed with 300 grams of potassium carbonate and 50 grams of ethylene glycol. The mixture is heated in carbondioxid atmosphere at a pressure of 8 atm. at. C. during 2 hours. The reaction product was cooled, then dissolved in water and neutralized by hydrochloric acid. The precipitate thus obtained is purified by dissolving in potassium carbonate and reprecipitation with hydrochloric acid. Yield: 82 grams of methylenedio-pyrocatechuic acid containin polycondensed products. Relative inhibitory activity: 3200.

What we claim is:

A compound selected from the group consisting' of compounds of the formula OOOH OOOH and its polycondensation products, all of which compounds are condensation products of gentisic acid with formaldehyde.

References Cited in the file of this patent UNITED STATES PATENTS Name Date Summers Aug. 5, 1902 Number OTHER REFERENCES 

